Most materials when exposed to high intensity radiation, particularly in the UV range, will naturally fluoresce for a period of time after the pulse of exciting radiation has subsided. Sometimes it is desirable, however, to record fluorescence of a fluorescent label present in a sample to be analyzed, where fluorescent radiation from the label must be distinguished from background fluorescent radiation emitted by other excited components in the sample. One technique in distinguishing background fluorescence from that of the fluorescing label is to make the label from a material which has a considerably longer fluorescing lifetime than the normal short life of background fluorescence. By conducting a time-resolved analysis of the fluorescence from the activated sample, the background fluorescence can be eliminated by allowing it to subside before analyzing for any fluorescent radiation from the label, if present in the sample.
This technique of analysis is very useful in a variety of diagnostic testing procedures. For example, the fluorescent label may be linked to a molecule which has an affinity for the molecule to be detected in the sample. An example of this technique is the common immunoassay. Time-resolved analysis of the fluorescent radiation is usually accomplished by a gating device, which is normally of electronic construction. Examples of such electronic gating devices are disclosed in U.S. Pat. Nos. 4,058,732; 4,198,567; and 4,612,660. Each of these patents discloses an electronic type of gating circuit which permits analysis of the fluorescent radiation after a predetermined delay from the time the sample was excited with a pulse of radiation directed on the sample.
Another form of gating system is disclosed in U.S. Pat. No. 4,336,459. This system is used to detect in daylight the presence of certain minerals in an uncovered ground sample. The gating may be accomplished by either an electronic actuated optic shutter or the shutter on a camera. One of the difficulties associated with this system is that the gated camera shutter cannot be opened and closed at sufficient speed to be applied in detecting fluorescence from a small concentration of fluorophores having shorter lifetimes of fluorescence.
In the fields of immunoassaying and DNA detection, there is significant demand for the ability to analyze for trace amounts of fluorescence. As is appreciated, fluorometry is a viable method in medical diagnostics, because it avoids the generally accepted hazardous radiation counting techniques. Background fluorescence continues to be one of the most significant problem in conducting such analysis. Background fluorescence can mask the radiation from the label and thereby confuse the test. Background fluorescence is emitted by many biological substances, such as blood serum, as well as solvents, plastics, glasses, papers and the like. These materials are commonly present in the medium or support for the sample to be analyzed. Another problem with fluorometry is the background interference caused by scattered radiation emitted by the excitation source. This also must be eliminated to improve accuracy of the test.
It has become possible to use fluorophores or labels which have a lifetime significantly longer than that of the background emissions from other components in the sample. Fluorescent rare earth chelates have lifetimes in the range of tens to thousands of microseconds, whereas the lifetime of the background fluorescence is normally in the range of tens to no more than hundreds of microseconds. The typical gated fluorometer of the type disclosed in the above patents, measures the intensity of the fluorescence starting after a predetermined delay time following the excitation pulse. This time-resolved approach allows the unwanted background emissions to subside to an insignificant level.
In medical diagnostics based on DNA fluorescent probing the testing is mostly qualitative. For this purpose time-resolved recording of the fluorescent image on a photographic film is sufficient.
Technically the problem is reduced to the question of how to photograph the fluorescence which is still being emitted after a specific time delay, usually hundreds of microseconds long, with respect to the excitation pulse. In other words, the opening of the shutter must occur after the given delay, and then the shutter has to open very rapidly that is, the process of its opening has to be not much longer than few hundreds of microseconds.
While the exposure time in modern conventional cameras can be as short as 250 .mu.s, the actual process of opening the shutter is much slower. This is because the very short exposures are accomplished by relatively slow movement, across the film surface, two curtains one following the other with a time delay equal to the exposure time. This means that, for example, for an exposure of 250 .mu.s, only a narrow slit of about 1 mm wide is travelling across the film. In effect the image of the object is recorded in strip by strip manner and if during the time of the slit sweep the object changes the intensity and wavelength composition of its emission, the photographic image will be recorded as changing its intensity and color also, along the direction of the slit movement. In conventional cameras it takes normally at least 10 milliseconds to complete one full sweep, which is definitely far too long if the recorded fluorescence has a lifetime of 1 ms. In such a case almost all the observed change of the fluorescence would be recorded on only 1/10 of the film frame length.
For the purpose of high speed photography streak cameras were developed. In this case, a very narrow slit placed in the optical path is swept across the film surface but at very high speed, comparable to the speed of the recorded event. As the event grows along the slit, it is recorded in graphical form with time in the one direction and position in the other.
The other photographic method used for recording fast changing events is high speed framing in which a series of pictures is taken covering the event. This is equivalent to the concept of a high speed motion picture camera.
There exist also the class of high speed camera shutters which are entirely non-mechanical. These include magneto- and electro-optical shutters, image intensifying tubes and electronic streak and framing cameras.